New paper: Unearthing diversity in fungal dark matter

To be born an orchid is a most unlikely thing. First your parents must be pollinated, which is difficult. Orchids are both rare, and rarely pollinated due to the bizarre and dishonest means by which they go about attracting pollinators. Added to that, orchids often rely on a single species of pollinator to do the job.

Let’s say, however, that your orchid parents do manage to achieve fertilization. Your orchid mother will produce many thousands of tiny dust-like seed, which will be jettisoned into the wind. Unlike most seeds, you have no maternal energy investment to power your germination and first days as a seedling. Instead, you must rely on blind luck to land you within reaching distance of a strand of soil fungus. This fungus is the wet nurse to bring you into the world, invading the seed coat and hooking the young orchid up to a network of fungal strands that pervade the soil. Tapping into this network provides you with the first sips of carbohydrate and nutrient you need in order to build your first green leaf and begin to stand on your own roots. But it is not enough to land near any fungus. Many orchid species require fungal partnership with a specific species of fungus for this to occur at all. Multiplied together, it is a wonder that orchids ever overcome these odds to propagate themselves into the next generation.

The southwest of Western Australia is rightly famous as a global biodiversity hotspot. The area is particularly rich in orchids, and the spider orchids (Caladenia) are some of the most impressive and diverse of the region’s main orchid groups. In 1967, University of Adelaide researcher John Warcup discovered in association with Caladenia a new genus of fungi. Today those fungi are called Serendipita, and although we have known of them for around 60 years, there have been less than a handful of species discovered and described.

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The spider orchid Caladenia arenicola was one of those sampled in the study

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White spider orchid (Caladenia splendens)

Ubiquitous yet invisible

Although related to mushrooms, Serendipita fungi have not been observed producing the conspicuous spore-bearing fruit bodies we usually use to find and identify them. This makes them largely invisible, and I have therefore never observed them in the wild. Despite that, recent research using DNA sequencing has found them to be absolutely everywhere. Inside all kinds of plants, outside all kinds of plants, and distributed from the equator to Antarctica. It is clear then that there must be a hidden biodiversity of these species siting, waiting to be discovered.

My study took a wide sample of southwest WA spider orchid samples and assayed them for the presence of Serendipita fungi. We then sequenced the DNA of all the fungi we found, and used a new analytical technique for dividing that DNA sequence diversity into units that are probably species. This is currently the only way to sensibly identify Serendipita fungi, as they all look completely alike and do not produce spores in the lab.

We found a total of eight species of Serendipita fungi, including the original species discovered by Warcup back in the 60s. These came from a total of 18 species of orchid. At some sites where we sampled multiple orchid species, we found six species of Serendipita, meaning that the fungi were as diverse as the orchids!

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Lying just below the soil horizon, that swollen, yellow stem bit is called the “collar”, and its where all spider orchids keep their fungus.

Untapped agricultural potential?

Although we have chosen to study these Serendipita in association with orchids, their wide host association has got other researchers interested in their role in plant health and application to agriculture. For example, Warcup’s species and one other have been used in experiments (and patent applications) showing inoculation with Serendipita results in profound benefits for the host plant, including:

  • Increased plant weight in maize, poplar, parsley, tobacco, barley, wheat, switchgrass and Arabidopsis
  • Enhanced grain yield in barley
  • Accelerated plant development in barley
  • Greater seed set, increased growth and faster flowering time in tobacco
  • Increased wheat yield in poor soils
  • Improved nutrient uptake in chickpea and lentil
  • Improved salinity tolerance in barley
  • Enhanced protection against root and stem pathogens in barley
  • Improved resistance to stem pathogens in tomato
  • Stronger defense response against mildew leaf pathogen in barley
  • Increased essential oil content in fennel and thyme
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Figure 7 from Ray and Craven (2016): Root growth in winter wheat in Serendipita vermifera inoculated plants (left) versus control (right)

These proven benefits make Serendipita a potentially powerful tool to enhance plant productivity and stress tolerance in crops. Furthermore, application of Serendipita fungi could be an organic alternative permitting growers to lower the application of unsustainable and ecologically harmful synthetic fertilizers. Our knowledge of plant-Serendipita associations in the wild suggests that these relationships are more prevalent in nutrient poor soils such as those in southwest WA. They are probably one factor that allows our plant diversity to thrive in such weathered, poor soils. This means that species of fungi that have evolved with the nutrient poor soils (like those discovered in this paper) might be untapped tools to enhance agriculture taking place in those very same soils.

 

(Erratum: This story was edited to replace the figure attributed to Ray and Craven (2016). The first image I used was one showing Arabidopsis capability for mycorrhizal association. Arabidopsis is typically thought to be a non-mycorrhizal plant, which is why this is interesting. The image however showed slower growth in the mycorrhizal treatment. A related Serendipita has been shown to enhance root growth in Arabidopsis however. I have now updated the post with a more appropriate image of root growth gains in wheat. Thanks to Pawel Waryszak (@PWaryszak) for pointing this out.)

 

My study:

Whitehead, M. R., Catullo, R. A., Ruibal, M., Dixon, K. W., Peakall, R., & Linde, C. C. (2017). Evaluating multilocus Bayesian species delimitation for discovery of cryptic mycorrhizal diversity. Fungal Ecology, 26, 74-84.

Further reading:

Weiß, M., Sýkorová, Z., Garnica, S., Riess, K., Martos, F., Krause, C., … & Redecker, D. (2011). Sebacinales everywhere: previously overlooked ubiquitous fungal endophytes. Plos one, 6(2), e16793.

Weiß, M., Waller, F., Zuccaro, A., & Selosse, M. A. (2016). Sebacinales–one thousand and one interactions with land plants. New Phytologist, 211(1), 20-40.

Ray, P., & Craven, K. D. (2016). Sebacinavermifera: a unique root symbiont with vast agronomic potential. World Journal of Microbiology and Biotechnology, 32(1), 16.

Bokati, D., & Craven, K. D. (2016). The cryptic Sebacinales: An obscure but ubiquitous group of root symbionts comes to light. Fungal Ecology, 22, 115-119.

Pollination, evolution and an orchid’s seductive ruse.

In a PR coup for dumpy little green orchids everywhere, research from my PhD recently landed on the cover of the journal Evolution. But what is it about?

Spring. The Blue Mountains, west of Sydney. Altitude 1000m. Frosty winds whip a swaying eucalypt canopy infiltrated by billowing cloud. Down below, amongst snowgrass tufts, rotting logs and bracken dwell the diminutive bird orchids. Genus: Chiloglottis. They huddle in tight colonies, sporadically sprayed by the high country squall.

Each plant holds two leaves pressed flat to the damp ground. Between the leaves a stem rises, holding aloft a single intricate flower in dusky shades of green and burgundy. When banks of cloud give way to azure sky and the shrike-thrushes resume their piping, these small blooms become irresistible lures.

Their target are the gracile flower wasps. Slim glossy black insects, zooming silently on shimmering wings. They are helplessly drawn to the flower. The bird orchid is emitting a scent, detectable only to wasps, which signals the promise of a mate. Known as ‘sexual deception’, the elaborate ruse uses a precise mimicry of female wasp pheromones to fool male wasps into pollinating the orchid.

However, here on the forest floor there is not only one species of orchid outwitting wasps for its own reproductive ends. Look closer and slight differences in the characteristics of flowers and visiting wasps betray something more complex and interesting. There are actually two species here, looking largely the same, growing in the same places, both deceiving their wasp pollinators through the false promise of sex.

By emitting subtle variations of their chemical trickery, these orchids have “tuned in” to two different pollinator species. This research paper explores this phenomenon as a way of separating the gene pools of closely related organisms. At the heart of it, the story here is about the forces that keep species apart once they split, or reproductive isolation.

First, we show that the different pheromones emitted by the two orchids are responsible for attracting different pollinators. Through arcane powers of chemical synthesis that I do not understand, chemists created synthetic orchid pheromones for us. We took these into the landscape and showed that the two chemicals attract two different wasps. The only perceivable difference between the wasps involved is yellow spangles on the carapace of one of the varieties. What’s more, this specific attraction is exclusive. Chemical A only attracts wasp A, and chemical B only appeals to wasp B.

Next, we take real flowers of both kinds and place them in a row and watch the hapless wasps roll in. We see that wasp A is only attracted to flower A, even when flower B is present just centimetres away. The results are identical to the results of the synthetic pheromone experiment.

On the basis of scent, we therefore expect that orchid A may never mate with orchid B. Exclusive attraction ensures that despite living amongst one another, some orchids may never exchange genes. Despite looking almost the same to us, they may as well exist on separate islands. They distinct separate species.

In order to back this up we then looked at the genetics of the species. By using the same kind of genes used in human DNA fingerprinting we were able to show that the two kinds of orchid exhibit differences in their gene pools of a degree expected if they were different species. Furthermore, analysis showed not a single individual displaying the genetics of a hybrid. Our last tests were to make hand-pollinated hybrids to check that hybrids could indeed form. These crosses showed hybrid offspring germinated and grew faster than pure crosses.

The potential for animals to drive the formation of plant species has long been recognized. This study gives us a strong case study of how that process might look. Our orchids are spectacular examples of the power of pollinators to create and maintain plant species. Through selective pollinator attraction, the orchids have been set upon unique and separate evolutionary journeys.

Further reading:

Whitehead, M. R. and Peakall, R. (2014) Pollinator specificity drives strong prepollination reproductive isolation in sympatric sexually deceptive orchids. Evolution 68: 1561–1575. doi: 10.1111/evo.12382

Rod Peakall and Michael R. Whitehead (2014) Floral odour chemistry defines species boundaries and underpins strong reproductive isolation in sexually deceptive orchids Annals of Botany 113 (2): 341-355 first published online September 19, 2013 doi:10.1093/aob/mct199